This project is to establish methods which allow one to target human genes in chicken B cell line,DT40, which has high homologous recombination activity. It has been reported that the exoginous DNA sequences integrate into homologous sequences in chicken genome by transfection of DT40 cells. If one can transfer a single copy of a human chromosome into DT40 and establish the monochromosomal hybrids, one might be able to target human genes in DT40. We transferred human chromosomes 1,2,3,11 and X from mouse A9 hybrids containing each of these chromosomes to DT40. DT40 cell lines containing human chromosomes 1,2,3 and 11 were successfully constructed. To test whether human genes can be targeted in DT40 hybrids by exoginous sequence, two gene loci, D11S16 locus on chromosome 11p13 and HRAS on chromosome 11p15 were chosen. Targeting constructs containing the D11S16 and HRAS sequences and zeomycin- resistant gene (mamalian selectable marker) were made and transfected to DT40 cells containing a human chromosome 11. Ten zeomycine-resistant clones from each transfection experiment were isolated. Southern blot analyses showed 60-70% of these clones were targeted, supporting the high frequency of a targeted integration of exoginous sequence. 1)We will generate DT40 hybrids containing human chromosomes 4 to 22. 2)We will generate chromosomal fragments from chromosome 1,3 and 11 for cellular senescence gene mapping, 3)We will knock-out the specific genes including mismatch repair genes hMSH2 on chromosome 2, hMLH1 on chromosome 3, and Ataxia Telangiectasia gene on chromosome 11 to determine the biological functions of these genes, 3) we will introduce a yeast selection marker to the specific gene loci to clone the specific regions into YACs, 4) we will introduce telomere sequence into specific gene loci to generate new chromosome with known deletion. These efforts will contribute to the mapping and cloning of novel cancer related genes.